Hereditary spherocytosis (HS), a common inherited hemolytic anemia, results from defects in the red blood cell (RBC) spectrin-based membrane skeleton, a multicomponent structure crucial to RBC mechanical stability. Defects in membrane skeleton components destabilize the membrane, leading to spherocytosis, splenic sequestration of damaged cells, and RBC dehydration. Heterogenous clinical presentation in humans results from both the specific protein defect and genetic diversity within the human population. An identical genetic lesion produces widely differing disease severity in human patients. Both the molecular interactions that underlie HS and the genetic loci that modulate its phenotype are ill defined. Deletion of the RBC integral membrane protein band 3 in mice causes profound HS without grossly affected membrane skeleton structure. Loss of the band 3 ligand, protein 4.2, produces mild HS and significantly alters RBC cation transport. Transfer of the band 3 null mutation to different inbred mouse strains lessens the severity of HS. The specific aims of this study test the hypotheses that (1) interactions between the membrane-spanning domain of band 3 and plasma membrane lipids stabilize the red cell membrane. The ability of the membrane-spanning and cytoplasmic domains of band 3 to independently stabilize the RBC membrane in vivo will be assessed using targeted mutagenesis in embryonic stem cells. The investigator will replace the endogenous band 3 gene (knockin) with a cDNA expressing: A) the band 3 transmembrane domain only, B) the band 3 cytoplasmic domain only, and C) the cytoplasmic domain of band 3 attached to the transmembrane domain of the polytopic RBC glucose transporter, Glut1; (2) interactions between protein 4.2 and cation transporters, or components of cation transport regulatory pathways, are critical determinants of RBC hydration status. The investigator will: A) replace the endogenous protein 4.2 gene with a protein 4.2 cDNA containing a mutated band 3 binding site, and B) identify novel protein 4.2 binding proteins in RBCs; (3) interactions between genetic loci significantly modulate RBC membrane characteristics and production. The investigator will genetically map the chromosomal location of genetic modifiers of the band 3 null HS phenotype, establish minimal genetic intervals containing the modifying loci, and build physical contigs spanning the minimal genetic intervals.